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N2 - A short synthesis of a hydroxyethylene dipeptide isostere, a core unit of the HIV protease inhibitors ritonavir and lopinavir, and its C-5 epimer is described.

DATA SYNTHESIS: Lopinavir/ritonavir is a fixed-dose protease ..

10/12/2016 · Kaletra (lopinavir/ritonavir)

The mechanisms by which lopinavir alters muscle protein synthesis have not been investigated previously. In general, regulation of protein synthesis involves changes in the phosphorylation state of several key components of the translation machinery including the phosphorylation of the elongation factor eEF2. Although we did not directly determine rates of elongation in the current study, these results are consistent with the observed reduction in protein synthesis in response to lopinavir. The effect of lopinavir on eEF2 is in agreement with previous studies examining various stressors. For example, treatment with the protease inhibitor indinavir decreased the activity of eEF2 in myocytes []. Likewise, alcohol or ATP depletion had a similar effect on the phosphorylation state of this elongation factor [; ].

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The elongation process consumes a considerable amount of cellular energy, thereby linking protein synthesis and signaling pathways that respond to changes in energy levels. For example, the AMPK pathway is activated in response to various stimuli that affect cellular energy levels. Furthermore, AMPK is known to regulate the activity of eEF2K [; ; ; ]. Thus, AMPK may be an important modulator of the effects of lopinavir on eEF2 phosphorylation. illustrates that lopinavir increased the phosphorylation of AMPK by 2-fold, when compared with control untreated cells. Lopinavir similarly increased the phosphorylation of ACC (), a known downstream substrate of AMPK. Both of these changes were independent of a change in total AMPK or ACC protein. Thus, these data are indicative of an overall increase in the activity of AMPK in myocytes after exposure to lopinavir.

DATA SYNTHESIS: Lopinavir/ritonavir …

Our studies also suggest that eEF2K is not necessarily required for the control of eEF2 phosphorylation. As such, treatment with the inhibitor rottlerin did not prevent the lopinavir-induced increase in eEF2 phosphorylation (), although it did suppress the stimulatory effect of lopinavir on eEF2K (). This idea is further supported by our in vitro kinase assay in which we used eEF2 as a substrate to directly measure lopinavir-induced changes in eEF2K activity (). This activity was blocked when rottlerin was included in the reaction mixture, verifying the ability of this drug to inhibit this step. These data are consistent with previous reports where rottlerin failed to block the stimulatory effect of alcohol on eEF2 phosphorylation, even though it did inhibit the increased activity of eEF2K in response to AICAR, FBS or growth factors [; ; ]. Thus, these results indicate that there is an alternative mechanism that can control eEF2 activity, without the involvement of eEF2K. This conclusion is in agreement with published studies whereby exercise or treatment with farnesyltransferase induced inactivation of eEF2 in association with inhibition of protein synthesis [; ]. These effects were also independent of the activity of eEF2K.

Kaletra (Lopinavir/Ritonavir) - Dec 04, 2016 - SAGE Pub

We propose a model in which lopinavir increases the phosphorylation and activity of AMPK, thereby leading to an increased phosphorylation and inactivation of eEF2. Based on our in vivo and in vitro inhibitor studies, we suggest that AMPK can act on eEF2 either directly, or indirectly via the action of eEF2K. For example, treatment with the inhibitor compound C blocks the ability of AMPK to phosphorylate either eEF2K or eEF2, although it does not directly inhibit the activity of eEF2K towards eEF2. Likewise, rottlerin treatments block the activity of eEF2K towards eEF2, without affecting AMPK. In summary, these data are in agreement with the decrease in protein synthesis that occurs in myocytes exposed to lopinavir. As such, this should provide insight into the AMPK signaling mechanism regulating this process.

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Previous in vitro and in vivo studies showed that various HIV protease inhibitors impaired protein synthesis, and this response was associated with defects in translation initiation and/or elongation. However, the effect that lopinavir has on these processes has not been reported. The aim of the present study was to determine whether lopinavir influences protein synthesis in C2C12 myocytes. In addition, we studied how proteins synthesis-related signaling events were regulated by this drug. Lopinavir decreased protein synthesis in a dose-and time-dependent manner, and this impairment was associated with an increase in eEF2 phosphorylation. Lopinavir also increased eEF2K phosphorylation and activity. Phosphorylation of eEF2K by lopinavir was mediated via activation of the AMPK pathway. On the other hand, in vitro kinase assays and studies using chemical inhibitors suggested that AMPK can regulate eEF2 independent of eEF2K.

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We studied the synthesis, cleavage rates, and oral administration of prodrugs of the HIV protease inhibitors (PIs) lopinavir and ritonavir. Phosphate esters attached directly to the central hydroxyl groups of these PIs did not demonstrate enzyme-mediated cleavage in vitro and did not provide measurable plasma levels of the parent drugs in vivo. However, oxymethylphosphate (OMP) and oxyethylphosphate (OEP) prodrugs provided improved rates of cleavage, high levels of aqueous solubility, and high plasma levels of the parent drugs when dosed orally in rats and dogs. Dosing unformulated capsules containing the solid prodrugs led to plasma levels equivalent to those observed for dosing formulated solutions of the parent drugs. A direct synthetic process for the preparation of OMP and OEP prodrugs was developed, and the improved synthetic method may be applicable to the preparation of analogous soluble prodrugs of other drug classes with limited solubility.

DATA SYNTHESIS: Lopinavir/ritonavir is a fixed-dose ..

HIV anti-retroviral drugs decrease protein synthesis, although the underlying regulatory mechanisms of this process are not fully established. Therefore, we investigated the effects of the HIV protease inhibitor lopinavir (LPV) on protein metabolism. We also characterized the mechanisms that mediate the effects of this drug on elongation factor-2 (eEF2), a key component of the translational machinery. Treatment of C2C12 myocytes with LPV produced a dose-dependent inhibitory effect on protein synthesis. This effect was observed at 15 min and was maintained for at least 4 h. Mechanistically, LPV increased the phosphorylation of eEF2 and thereby decreased the activity of this protein. Increased phosphorylation of eEF2 was associated with increased activity of its upstream regulators AMP-activated protein kinase (AMPK) and eEF2 kinase (eEF2K). Both AMPK and eEF2K directly phosphorylated eEF2 in an in vitro kinase assay suggesting two distinct paths lead to eEF2 phosphorylation. To verify this connection, myocytes were treated with the AMPK inhibitor compound C. Compound C blocked eEF2K and eEF2 phosphorylation, demonstrating that LPV affects eEF2 activity via an AMPK-eEF2K dependent pathway. In contrast, incubation of myocytes with rottlerin suppressed eEF2K, but not eEF2 phosphorylation, suggesting that eEF2 can be regulated independent of eEF2K. Finally, LPV did not affect PP2A activity when either eEF2 or peptide was used as the substrate. Collectively, these results indicate that LPV decreases protein synthesis, at least in part, via inhibition of eEF2. This appears regulated by AMPK which can act directly on eEF2 or indirectly via the action of eEF2K.